Overexpressed DNA methyltransferase 1 (DNMT1) strongly contributes to tumor suppressor gene silencing in colorectal cancer (CRC). However, the underlying mechanism of DNMT1 overexpression is still unclear. MicroRNAs (miRNA) have been implicated as gene regulators controlling diverse biological processes, including carcinogenesis. In this study, we investigated whether some miRNA is involved in the regulation of DNMT1 and thus play a functional role in CRC. Our results showed that miR-342 was downregulated in CRC tissues and cell lines. Restoration of miR-342 resulted in a dramatic reduction of the expression of DNMT1 at both messenger RNA and protein levels by directly targeting its 3' untranslated region. This in turn reactivated ADAM23, Hint1, RASSF1A and RECK genes via promoter demethylation. Furthermore, the enhanced expression of miR-342 could significantly inhibit SW480 cell proliferation in vitro (P = 0.006). Further investigation demonstrated G(0)/G(1) cell cycle arrest in SW480 cells, which was associated with an upregulation of p21 and downregulation of cyclinE and CDK2. Overexpression of miR-342 also inhibited SW480 cell invasion. The in vivo antitumor effect was evaluated in SW480 cells with lentivirus-mediated expression of miR-342. Results showed that overexpression of miR-342 significantly inhibited tumor growth and lung metastasis in nude mice (P = 0.034). Our findings describe a new mechanism for the regulation of DNMT1 and aberrant DNA hypermethylation in CRC. This is also the first report to demonstrate that miR-342 may act as a tumor suppressor gene in CRC development. The newly identified miR-342/DNMT1 link provides a new, potential therapeutic target for the treatment of CRC.

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BackgroundHypoxic tumor cells can reduce the efficacy of radiation. Antiangiogenic therapy may transiently “normalize” the tumor vasculature to make it more efficient for oxygen delivery. The aim of this study is to investigate whether the recombinant human endostatin (endostar) can create a “vascular normalization window” to alleviate hypoxia and enhance the inhibitory effects of radiation therapy in human nasopharyngeal carcinoma (NPC) in mice.Methodology/Principal FindingsTransient changes in morphology of tumor vasculature and hypoxic tumor cell fraction in response to endostar were detected in mice bearing CNE-2 and 5–8F human NPC xenografts. Various treatment schedules were tested to assess the influence of endostar on the effect of radiation therapy. Several important factors relevant to the angiogenesis were identified through immunohistochemical staining. During endostar treatment, tumor vascularity decreased, while the basement membrane and pericyte coverage associated with endothelial cells increased, which supported the idea of vessel normalization. Hypoxic tumor cell fraction also decreased after the treatment. The transient modulation of tumor physiology caused by endostar improved the effect of radiation treatment compared with other treatment schedules. The expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14 decreased, while the level of pigment epithelium-derived factor (PEDF) increased.ConclusionsEndostar normalized tumor vasculature, which alleviated hypoxia and significantly sensitized the function of radiation in anti-tumor in human NPC. The results provide an important experimental basis for combining endostar with radiation therapy in human NPC.

The aim of this study was to investigate the effect of microRNA-20a on pancreatic carcinoma cell proliferation and invasion and to find a new effective treatment strategy for pancreatic carcinoma. MicroRNA-20a expression was determined in 10 matched normal pancreatic tissues and pancreatic carcinoma by in situ hybridization. Quantitative real-time RT-PCR was used to evaluate the expression of microRNA-20a in two pancreatic carcinoma cell lines (BxPC-3 and Panc-1) and immortal human pancreatic duct epithelial cell line H6C7. Proliferation and invasion capacity were analyzed for the cells with lentivirus-mediated overexpression of microRNA-20a both in vitro and in vivo. In addition, the regulation of signal transducer and activator of transcription proteins 3 (Stat3) by microRNA-20a was determined to elucidate the underlying mechanisms. The pancreatic cancer cell lines (Panc-1 and BxPC-3) stably overexpressing microRNA-20a showed reduced proliferation and invasion capacity in vitro and in vivo, compared with parental cells or cells transfected with a control vector. Furthermore, we found that microRNA-20a negatively regulated Stat3 protein expression in a dose-dependent manner without changing the Stat3 mRNA level and decreased the activity of a luciferase reporter construct containing the Stat3 3'-untranslated region. These results show that microRNA-20a regulates Stat3 at the post-transcriptional level, resulting in inhibition of cell proliferation and invasion of pancreatic carcinoma. It may open a new perspective for the development of effective gene therapy for pancreatic carcinoma.

Nasopharyngeal carcinoma (NPC), which has the highest incidence in South China, is mainly treated by radiotherapy. However, the survival rate remains low. Angiogenesis is closely correlated with progress of NPC. Thus, the combination of anti-angiogenesis with radiation is an attractive strategy for NPC treatment. A heterogenic xenografted human NPC nude mice model was established to investigate the effect of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic factor, and the combined effect of PEDF and radiotherapy on nasopharyngeal carcinoma. Pigment epithelium-derived factor remarkably suppressed the growth of NPC by 43.52% and decreased the tumor microvessel density (MVD). Pigment epithelium-derived factor had no effects on the proliferation and apoptosis of NPC cell lines by MTT and flow cytometry assay. However, PEDF decreased vascular endothelial growth factor (VEGF) in NPC cell lines by downregulation of hypoxia-inducible factor 1a, a crucial transcriptional factor for VEGF expression, as demonstrated by western blotting and immunofluorescent staining assay. Interestingly, irradiation alone could also effectively downregulate VEGF and MVD of xenografted tumor, which indicates that irradiation suppresses NPC not only by killing tumor cells but also through anti-angiogenesis. Furthermore, combined treatment of PEDF with irradiation enhanced the antitumor efficacy. The MVD and VEGF in the combined therapy were much less than in the treatment with PEDF or radiotherapy alone. Our observation demonstrated that the combination of PEDF with radiotherapy enhances the efficacy of the antitumor effect on NPC by the coordinated inhibition on angiogenesis, which implies the potential role of PEDF as an adjuvant agent for NPC treatment. (Cancer Sci 2011; 102: 1789-1798 N asopharyngeal carcinoma (NPC) is rare in most countries, with an age-adjusted incidence less than one per 100 000 population per year. However, the highest incidence is found in Southern China (25-30 per 100 000 population per year), especially among people of Cantonese ancestry.(1) Nasopharyngeal carcinoma has the highest incidence of distant metastasis among head and neck cancers.(2,3) Radiotherapy is the main strategy for NPC. With the improvement of modern imaging techniques and radiotherapy planning and delivery, the localregional control of this neoplasm has been improved. (4,5) Overall survival has also been improved with the addition of concurrent and adjuvant chemotherapy.(6,7) However, the incidence of relapse remains high. Therefore, the development of multidisciplinary therapeutic approaches to improve local-regional control and eradicate micrometastases is required. (8) Angiogenesis, the formation of new blood vessels from preexisting vessels, occurs physiologically in growth, development and pathologically in tumors, diabetic retinopathy, rheumatoid arthritis and regeneration.(9) Tumor growth and metastasis are angiogenesis dependent, therefore inhibiting tumor-induced angiogenesis is a promising strategy for the treatment...

BackgroundNonviral vectors are attractively used for gene therapy owing to their distinctive advantages. Our previous study has demonstrated that transfer of human IFNγ gene into nasopharyngeal carcinoma (NPC) by using a novel nonviral vector, minicircle (mc), under the control of cytomegalovirus (CMV) promoter was effective to inhibit tumor growth. However, therapies based on CMV promoter cannot express the targeted genes in cancer tissues. Previous studies indicated that the development of human NPC was closely associated with Epstein-Barr virus (EBV) and demonstrated the transcriptional enhancer function of oriP when bound by EBV protein. Therefore, the present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNγ) in which the transgene expression was under the transcriptional regulation of oriP promoter.Methodology/Principal FindingsDual-luciferase reporter assay and ELISA were used to assess the expression of luciferase and IFNγ. WST assay was used to assess the cell proliferation. RT-PCR was used to detect the mRNA level of EBNA1. RNAi was used to knockdown the expression of EBNA1. NPC xenograft models in nude mice were used to investigate the targeted antitumor efficacy of mc-oriP-IFNγ. Immunohistochemistry was used to detect the expression and the activity of the IFNγ in tumor sections. Our results demonstrated that mc-oriP vectors mediated comparable gene expression and anti-proliferative effect in the EBV-positive NPC cell line C666-1 compared to mc-CMV vectors. Furthermore, mc-oriP vectors exhibited much lower killing effects on EBV-negative cell lines compared to mc-CMV vectors. The targeted expression of mc-oriP vectors was inhibited by EBNA1-siRNA in C666-1. This selective expression was corroborated in EBV-positive and -negative tumor models.Conclusions/SignificanceThis study demonstrates the feasibility of mc-oriP-IFNγ as a safe and highly effective targeted gene therapeutic system for the treatment of EBV positive NPC.

Background:There is mounting evidence that microRNAs play an important role in nasopharyngeal carcinoma, which is widely prevalent in South China and is the most prevalent metastatic cancer among head and neck cancers. Recently, it has been shown that miR-494 is involved in the progression and prognosis of nasopharyngeal carcinoma. However, little is known about the function and mechanism of miR-494-3p in nasopharyngeal carcinoma. In the present study, we aimed to investigate the effects of miR-494-3p on the migration and invasion of nasopharyngeal carcinoma and to further explore the underlying mechanisms of these processes.Methods:The expression levels of miR-494-3p and Sox7 in nasopharyngeal carcinoma specimens and nasopharyngeal carcinoma cell lines were measured using quantitative reverse transcription polymerase chain reaction. Luciferase reporter assay, quantitative reverse transcription polymerase chain reaction, and Western blotting were used to confirm whether Sox7 was a direct target of miR-494-3p. Additionally, the roles of miR-494-3p and Sox7 on cell proliferation, migration, and invasion of nasopharyngeal carcinoma were analyzed by Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and Boyden chamber assay, respectively.Results:Our study demonstrated that miR-494-3p was commonly upregulated in nasopharyngeal carcinoma specimens and nasopharyngeal carcinoma cell lines compared with nontumor nasopharyngeal epithelial tissue or nasopharyngeal cells (NP69). Moreover, miR-494-3p negatively regulated Sox7 at the posttranscriptional level by binding to a specific site in the Sox7 3′-untranslated region. In addition, synthetic miR-494-3p mimics significantly promoted proliferation, migration, and invasion of S18 and S26 nasopharyngeal carcinoma cells, while a synthetic miR-494-3p inhibitor resulted in suppressed nasopharyngeal carcinoma cell migration and invasion.Conclusion:miR-494-3p promotes nasopharyngeal carcinoma cell growth, migration, and invasion by directly targeting Sox7. Our results suggest that miR-494-3p might be a potential therapeutic target for nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a leading cause of cancer-related mortality. Radiotherapy is one of the primary modalities for NPC treatment. However, in patients in the late stages of the disease, the local control rate and overall survival rate remain low. Therefore, it is urgent to identify new targets that can improve the outcome of radiotherapy in this neoplasm. In the present study, we investigated the effects of metformin on the radiosensitivity of NPC cells and explored the potential mechanisms. The radiosensitizing effects of metformin on NPC cells were measured by colony formation assay. Cell apoptosis was assessed by Hoechst 33342 staining analysis. DNA damage was detected by monitoring γ-H2AX foci with immunofluorescence. The changes in apotosis-related and DNA damage repair-related proteins were detected by western blotting. Our study demonstrated that metformin significantly reduced the cell viability, enhanced radiosensitivity and potentiated radiation-induced caspase-9/-3 cleavage in the NPC cells. In addition, metformin plus radiation significantly upregulated the expression of p-ATM, p-ATR, γ-H2AX and downregulated the expression of ATM, ATR, p95/NBS1, Rad50, DNA-PK, Ku70 and Ku80. Therefore, our results suggest that metformin possesses a strong radiosensitizing potential in NPC cells. This radiosensitizing effect was associated with inhibition of DNA double-strand break repair processes through HR repair and the NHEJ repair signaling pathway, thereby enhancing radiation-induced cell apoptosis. These findings imply that metformin is a potent radiation-sensitizing agent and may be a promising candidate for clinical evaluation as part of a combined regimen for the treatment of nasopharyngeal carcinoma.